PCR (polymerase chain reaction) and other techniques to amplify particular nucleic acids are applied in various fields in biotechnology. (Hereinafter, throughout this specification, these PCR and other nucleic acid amplification reactions will be referred to as “nucleic acid amplification reactions.”)
A nucleic acid amplification reaction like PCR generally requires a process of confirming whether a target nucleic acid has been specifically amplified. (Amplification of nucleic acid by PCR or a like nucleic acid amplification method will be simply referred to as “nucleic acid amplification” throughout this specification.)
Amplification of nucleic acid is confirmed, for example, as follows: The reaction liquid having undergone PCR or a like nucleic acid amplification reaction is subjected to get electrophoresis using polyimide or another like gel. The DNA fragments obtained by the PCR amplification are then stained.
Nucleic acid amplification in a nucleic acid amplification reaction can be confirmed also by other conventional methods. For example, the turbidity of the reaction liquid having undergone a nucleic acid amplification reaction may be measured. A microarray may be used including a probe which binds specifically to target nucleic acid. Another example is real time PCR by which the amplification can be confirmed in real time by using a fluorescent-labelled probe which binds to a double-stranded DNA or specifically to a target PCR product.
The nucleic acid amplification reaction, including PCR, is used also, for example, to analyze single nucleotide polymorphisms (hereinafter, “SNPs”). The methods above are also used in these cases to confirm nucleic acid amplification.
Patent literature 1 proposes a primer for use with wildtype and one or two kinds of primers for use with mutation type simultaneously or separately acting, together with a DNA polymerase, on a chromosome or its fragment which includes an SNP site to be analyzed, in order to find if any primer-based extension has occurred. Electrophoresis is used in the analysis to confirm presence of an amplified nucleic acid.
Patent literature 2 suggests use of a universal primer and two kinds of specific primers, one for use with a reference sequence and the other for use with a mutated sequence, both of which include an SNP site, in order to amplify a target part of a sequence. This SNP analysis, similarly to patent literature 1, confirms presence of amplified product by subjecting the reaction liquid obtained to electrophoresis.
Patent literature 3 suggests use of target genome DNA and pairs of primers, in order to amplify a nucleic acid which includes an SNP site for typing. The amplified product obtained is hybridized using, for example, a labelled probe for the typing.
Quick and convenient SNP analysis would enable tailor-made medical care (for example, bedside diagnostics for the best therapy and drug administration), making promising POC (Point Of Care) technology. To achieve these goals, a method is desired which can more quickly and more conveniently confirm amplification of a nucleic acid after a nucleic acid amplification reaction.